F23 Lab 7 Report
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BIOL 3380
Experiment 7 - Lab Report
Western Blot Analysis of rGFP Fractions
Name:
Rania Mohamed
Circle your lab section:
005
T–AM
W-AM
R-AM
F-AM
T–PM
W-PM
R-PM
F-PM
Instructor:
Mazambani Simarashe
Graduate TA:
Falah Rafah
Date:
11/15/2023
Partner:
Varshini
7-
1
Experiment 7 – Lab Report
Western Blot Analysis of rGFP fractions
Simply edit this word document by inserting your answers after each question/space. Please
denote your typed answers by using
bold
,
italic
s, or
colored
font
. Do NOT renumber/reorganize
the questions.
“Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of
your handwritten work, or using handwritten annotation on the document (if you have the
technology to do so).
1.
(10pts) You will receive these points if you performed the SDS-PAGE and the Western
Transfer correctly.
2.
(2pts) What is the GENERAL purpose of a Western Blot?
Detect presence of rGFP by using a specific antibody
3.
(2pts) Ponceau S is a red dye that reversibly binds to all proteins. We used this dye to stain
the membrane prior to developing the Western blot with antibodies.
One student’s blot did
not have any bands after performing the Ponceau S stain.
Describe one likely
procedural
error during the transfer set-up
that would account for this observation.
The protein side of the membrane was facing down instead of upside and so the
dye did not bind to it
4.
(2pts) Why did we use a primary antibody that recognizes the Xpress epitope tag and not the
His
6
tag to detect rGFP?
The his6 tag could have bound other contaminants besides rGFP but
xpress epitope tag is more specific for the antibody
5.
(5pts) Make a figure of your Western blot result with appropriate titles and labels.
Please see page 7-4
6.
(2pts) Describe the data from the G0, G3, and GCE lanes of your Western blot result. Are
those results expected? Explain why or why not.
G0 had no bands. G3 and GCE lanes had bands in them. These results were
expected because at G0 there was no IPTG while G3 was induced with
IPTG and rGFP was expressed
7.
Based on your knowledge of Experiments 3-7, when you developed the Western blot:
a.
(1pt) What MW band in your elution lanes are you expecting to observe in the final
result?
About 32 kD
7-
2
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Following the publication of the genetic sequence of SARS-CoV-2 on January 11, 2020, intense research efforts have been devoted to developing a vaccine against COVID-19. With unprecedented speed, this extraordinary scientific mobilization led the first vaccine candidate to enter the Phase I human clinical trial on March 16, 2020, and other novel candidates are rapidly following. Up to May 22, 2020, there are 10 COVID-19 candidate vaccines in clinical evaluations and 114 in preclinical development.
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Summary
Grades for Arysta Visser: 23 x M Uh-oh! There's a problem w X b The restriction EcoRI cleaves X + Untitled spreadsheet - Goog X
https://docs.google.com/document/d/1mKY1HIgMPRh1kRDCmX7msBF2yf07-ogT/edit
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18. The restriction EcoRI cleaves double-stranded DNA at the sequence 5'-GAATTC-3', the
restriction enzyme HindIII cleaves at the sequence 5'-AAGCTT-3', and the restriction enzyme
BamHI cleaves at 5'GGATCC-3. An 805 bp circular plasmid is digested with each enzyme
individually and then in combination, and the resulting fragment sizes are determined by
means of electrophoresis. The results are as follows:
1
Restriction Enzyme(s)
EcoRI
BamHI
HindIII
EcoRI and BamHI
EcoRI and HindIII
BamHI and HindIII
3
Practice
====•=•€ EX
Fragment lengths (base pairs)
430 bp, 375 bp
470 bp, 335 bp
Lab Report 6…
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POST-LAB QUESTIONS
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Biotechnology 165
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